Processes for separation of sterol compounds from fluid mixtures using substantially insoluble compounds

ABSTRACT

Cholesterol and other sterols are separated from fluid mixtures, especially foodstuffs such as egg yolk, using a surface-modified substantially insoluble carbonate salt which selectively adsorbs the sterol. The surface-modified carbonate is prepared by (a) treating a substantially insoluble carbonate salt with a sterol compound so that the sterol compound becomes adsorbed on the carbonate surface; (b) treating the sterol-modified carbonate with a surface-modifying agent, said surface-modifying agent having a reactive group capable of reacting with the surface of the carbonate, and an elongate hydrophobic portion, so that the surface of the carbonate not covered by the adsorbed sterol compound reacts with the surface-modifying agent; and (c) desorbing the sterol compound from the carbonate. A surface-modified carbonate prepared in this way may also be used to remove sterols from solvents (such as carbon dioxide) which have themselves been used to extract sterols from foodstuffs, thus avoiding the need to distil and condense the solvent before it is recycled to treat further batches of the foodstuff.

FIELD OF THE INVENTION

This invention relates to processes for separation of sterol compounds(a term which is used herein to refer to cholesterol and itsderivatives, metabolites and enzymatic degradation products, and alsoincludes plant sterols and the oxidized forms of such plant sterols)from the fluid mixtures using substantially insoluble compounds, namelysubstantially insoluble carbonate salts. The process is especially,though not exclusively, useful for the separation of sterol compoundsfrom egg yolks, but may also be used to separate such compounds fromfish oil, butter oil, lard, tallow and other fatty materials.

BACKGROUND OF THE INVENTION

Cholesterol and other sterols are natural constituents of manyfoodstuffs. However, the presence of large amounts of cholesterol andother sterols in the human body is considered by physicians to bedeleterious, since cholesterol has been implicated as a factor in anumber of diseases, especially atherosclerosis, in which depositscontaining a high proportion of cholesterol are deposited in bloodvessels. Accordingly, it is common practice for physicians to recommendto patients who have suffered a heart attack, or who display alikelihood of, or documented, hypercholesterolemia, that the patientsreduce their cholesterol intake from foodstuffs.

However, cholesterol is found in significant quantities in a widevariety of foodstuffs, being present in most animal fats, andconsequently restrictions upon the cholesterol intake of patientsnecessitate prohibiting or greatly reducing the consumption of manyfoodstuffs, a step which many patients are reluctant to take, and whichmay introduce complications in ensuring that the patients receive aproperly balanced diet meeting all nutritional requirements. Moreover,cholesterol is present in large amounts in egg yolks. Eggs are used insome processed food applications and formulas as a binding agent, and ifthe eggs are eliminated it is difficult to produce a foodstuff havingthe expected texture. Finally, the public has recently becomeincreasingly aware of the health risks associated with consumption ofcholesterol, so that even persons who are not under medical treatmentfor conditions in which cholesterol is implicated are voluntarilyattempting to reduce their cholesterol consumption, and the foodindustry is beginning to label foods to show their cholesterol content.Thus, many people may avoid foods known to be high in cholesterol andseek substitutes.

In order to help people to reduce their cholesterol consumption withoutmajor modifications in their diet (and thus help to ensure, inter alia,that people who must follow a low cholesterol diet for medial reasons doin fact keep to such a diet), it is desirable to provide some method bywhich cholesterol and other sterol compounds (many of which can bemetabolized to cholesterol or its derivatives) can be extracted fromvarious foodstuffs, thereby producing low-cholesterol versions of suchfoodstuffs which can be consumed in place of the original,high-cholesterol foodstuffs. However, the requirements for such asterol-removal process are exacting. The process must not, of course,introduce into the foodstuff any material which is not generallyrecognized as safe for use in foodstuffs. The process should remove fromthe foodstuff not only cholesterol itself but also cholesterolderivatives and other sterol compounds which can be metabolized in thebody to cholesterol or derivatives thereof, and which thus affectcholesterol levels in the body. Furthermore, the process should leavethe foodstuff in a form which is as close as possible to that of theoriginal, high-cholesterol foodstuff. For example, when used on eggyolks, the cholesterol-removal process should maintain the highviscosity of the egg yolks, since this high viscosity is needed forproper binding action when the treated egg yolk is used in theproduction of baked goods, liquid egg substitutes, margarines etc., andshould not denature the proteins present in the egg yolk. Finally, thecholesterol-removal process should preserve the nutritional value of thefoodstuff, and not, for example, remove vitamins and other importantconstituents of the foodstuff. In particular, since cholesterol isfrequently present in foodstuffs in the form of various complexes, it isdesirable that a cholesterol-removal process not remove the othernatural materials found to be associated with the cholesterol.

Numerous attempts have previously been made to provide acholesterol-removal process which meets these exacting criteria. Forexample, attempts have been made to remove cholesterol, and otherundesirable food components, by extracting the cholesterol from thefoodstuff with liquid, usually supercritical, carbon dioxide. Suchcarbon dioxide extraction processes suffer from the disadvantage thatthey must be operated under pressure to keep the carbon dioxide in theliquid phase, which increases the cost of the apparatus required. Inaddition, such carbon dioxide extraction processes may not be veryselective in removing cholesterol, and thus may remove valuableconstituents of the foodstuff. In addition, the properties of somefoodstuffs may be altered disadvantageously by contact with liquidcarbon dioxide; for example, in some cases the carbon dioxide may removeflavoring and/or odiferous components, thereby affecting the tasteand/or smell of the treated foodstuff.

For example, U.S. Pat. No. 4,692,280, issued Sept. 8, 1987, to Spinelliet al., describes a process for the purification of fish oils in whichthe oil is extracted with supercritical carbon dioxide to removecholesterol, together with odoriferous and volatile impurities.

Food Science and Technology Abstracts, 6, Abstract 8 A 374 (1974)(Abstract of Food Technology 28(6), 32-34, 36, 38 (1974)) describes apilot plant for extraction of volatile substances from liquid and solidfoods using liquid carbon dioxide as the solvent.

Food Science and Technology Abstracts, 18(1), Abstract 1 H 46 (1986)(Abstract of German Offenlegungsschrift 33 31 906 (1985)) describesextraction of caffeine from coffee beans using supercritical carbondioxide as the solvent.

Food Science and Technology Abstracts, 18(2), Abstract 2 M 118 (1986)(Abstract of Agricultural and Biological Chemistry, 49(8), 2367-2372(1985)) describes extraction of oils from wheat germ using supercriticalcarbon dioxide as the solvent.

Food Science and Technology Abstracts, 18(3), Abstract 3 T 70 (1986)(Abstract of Indian Food Industry, 3(2), 48-51 (1084)) describesextraction of flavor components from natural products using both liquidand supercritical carbon dioxide.

Food Science and Technology Abstracts, 18(8), Abstract 8 V 321 (1986)(Abstract of French Patent Application Publication No. 2,563,702 (1985))describes extraction of essential oils from blackcurrant buds usingsupercritical carbon dioxide.

Food Science and Technology Abstracts, 19(3), Abstract 3 V 102 (1987)(Abstract of United Kingdom Patent Application Publication No. 2,173,985(1986)) describes extraction of aroma materials from dried plantmaterial, which has been milled and soaked in ethanol, using acontinuously flowing stream of carbon dioxide at a temperature below itscritical temperature. The plant material can be used for extraction oftannin, caffeine and nicotine from tea, coffee and tobacco respectively.

Food Science and Technology Abstracts, 19(4), Abstract 4 E 11 (1987)(Abstract of Food Manufacture, 61(12), 58 (1986)) describes the use ofliquid, supercritical or high pressure carbon dioxide in variousprocesses, including decaffeination of coffee, preparation of hopextract for brewing, extraction of essential oils, defatting of potatochips, and fractionation of fish oils.

Food Science and Technology Abstracts, 19(4), Abstract 4 N 36 (1987)(Abstract of Seafood Export Journal 18(9), 10-13 (1986)) describesextraction of oils from Antarctic krill using supercritical carbondioxide.

Food Science and Technology Abstracts, 19(6), Abstract 6 G 29 (1987)(Abstract of Nahrung 30(7), 667-671 (1986)) describes defatting ofbaker's yeast protein extracts by extraction with supercritical carbondioxide.

Food Science and Technology Abstracts, 19(12), Abstract 12 H 200 (1987)(Abstract of Journal of Food Science and Technology 23(6), 326-328(1986)) describes decaffeination of coffee using supercritical carbondioxide as solvent.

Food Science and Technology Abstracts, 20(2), Abstract 2 E 35 (1988)(Abstract of Voedingsmiddelentechnologie 20(7), 32-35 (1987)) describesvarious uses of extraction with supercritical carbon dioxide in the foodindustry, including extraction of oils and fats, preparation of hopextracts, fractionation of oils and fats, extraction of essential oils,and elimination of undesirable constituents, for example decaffeinationof coffee.

Food Science and Technology Abstracts, 20(3), Abstract 3 N 31 (1988)(Abstract of Agricultural and Biological Chemistry, 51(7), 1773-1777(1987)) describes fractional extraction of rice bran oil withsupercritical carbon dioxide.

Food Science and Technology Abstracts, 20(4), Abstract 4 E 36 (1988)(Abstract of Food Trade Review 57(9), 461, 463-464 (1987)) describes theuse of supercritical carbon dioxide as an extractant of vegetable oils.

Swientek, Supercritical fluid extraction separates components in foods,Food Processing 48(7), 32, 34, 36 (1987)) describes the use ofsupercritical fluid extraction in the food industry, including removalof cholesterol from milkfat, extraction of omega-3-fatty acids from fishoil, and extraction of oil seeds.

Food Science and Technology Abstracts, 20(5), Abstract 5 T 58 (1988)(Abstract of Sciences des Aliments 7(3), 481-498 (1987)) describes thepreparation of a black pepper oleoresin by extraction of the pepper withsupercritical carbon dioxide or with a carbon dioxide/ethanol blend.

Food Science and Technology Abstracts, 20(6), Abstract 5 T 58 (1988)(Abstract of West German Patentschrift 30 11 185 (1988)) describes thepurification of lecithin for food or pharmaceutical use by extractionwith supercritical carbon dioxide.

Food Science and Technology Abstracts, 20(7), Abstract 7 N 60 (1988)(Abstract of Journal of the American Oil Chemists' Society 65(1),109-117 (1988)) describes fractionation of menhaden oil ethyl estersusing supercritical fluid carbon dioxide to produce cholesterol-rich andcholesterol-depleted fractions.

Food Science and Technology Abstracts, 20(8), Abstract 8 E 4 (1988)(Abstract of Bio/Technology 6(4), 393-394, 396 (1988)) describesindustrial scale use of supercritical fluid extraction with retrogradecondensation to recover the condensation to recover the solute.Applications of this technology include extraction of caffeine fromcoffee, removal of toxic thujone from wormwood flavoring, extraction oftriacylglycerols from many sources, extraction of sterols and steroidsfrom poultry and meat products, and extraction of essential oils fromthyme.

Food Science and Technology Abstracts, 20(8), Abstract 8 N 26 (1988)(Abstract of Energy in Agriculture 6(3), 265-271 (1987)) describesextraction of peanut oil using supercritical carbon dioxide.

Food Science and Technology Abstracts, 20(12), Abstract 12 N 16 (1988)(Abstract of Dissertation Abstracts International, B 48(9), 2632 (1988))describes extraction of oil from Canola (Brassica napus or B.campestris) seed using supercritical carbon dioxide.

In addition to the problems previously mentioned, prior art processesfor extraction of cholesterol and other components from foodstuffs usingliquid or supercritical carbon dioxide normally involve high energycosts, since not only is the carbon dioxide itself costly, but beforethe carbon dioxide can be recycled to treat further batches of thefoodstuff, the dissolved cholesterol is removed by allowing the carbondioxide to evaporate (technically speaking, supercritical carbon dioxideis simple decompressed) to produce gaseous carbon dioxide and a liquidor solid residue, and the gaseous carbon dioxide must then berecompressed (and if necessary liquified) to produce liquid orsupercritical carbon dioxide; this recompression is energy intensive.Accordingly, the cost of extraction of cholesterol from foodstuffs usingliquid or supercritical carbon dioxide could be reduced if a way couldbe found to remove cholesterol from the carbon dioxide without the needto evaporate and recompress this material. This invention provides sucha process for removal of cholesterol or other sterol compounds fromcarbon dioxide or other solvent laden with these sterols.

Furthermore, a wide variety of techniques have previously been employedin the extraction of materials from, and the purification of, complexorganic mixtures, and examples of such techniques will now be given.

Food Science and Technology Abstracts, 20(11), Abstract 11 V 36 (1988)(Abstract of International Patent Application Publication No. WO88/02989 (1988)) describes a process for the simultaneous deodorizationand cholesterol reduction of fats and oils by deaeration, mixing withsteam, heating, flash vaporizing, thin-film stripping withcountercurrent steam, and cooling (all the preceding steps beingperformed under vacuum), and storage under oxygen-free conditions. Thisprocess demonstrates the difficulty in removing cholesterol from afoodstuff while maintaining the expected flavor thereof.

Deutsch et al., "Isolation of Lipids from plasma by AffinityChromatography", Biochemical and Biophysical Research Communications,50(3), 758-764 (1973) describes the extraction of certain lipidfractions from plasma by affinity chromatography. Cross-linked agarose(SEPHAROSE 4B) was activated by the well known cyanogen bromide methodof Cuatrecasas. Dodecylamine was then covalently bound to the activatedagarose to provide the adsorbent. Plasma was mixed with the adsorbent,whereupon the adsorbent was then filtered and washed. Lipids were theneluted off the adsorbent with ethanol. This procedure removedapproximately 50% of the triglycerides and nearly all of the cholesteroland lipoproteins. This procedure of would be expected to work on dairyproducts such as milk. However, milk products require triglycerides andlipoproteins for integrity, mouthfeel and taste. For example, the fat inbutter is necessary for cooking. Accordingly, this procedure would altermilk products such that much of the fat and nutrients would be removed.

U.S. Pat. No. 4,431,544 to Atkinson et al. teaches a high pressureliquid affinity chromatography by which biomolecules are extracted fromsolution and purified. Ligands are attached to matrices by way of spacerarms to provide the adsorbent. The matrix may be cross-linked agarose,and the spacer arms may be polyarginine or polylysine. The extraction ofcholesterol from dairy products through this general-ligand affinitychromatography process is not feasible because of the broad specificity,and the toxic nature of the crosslinking agents. For example, cyanogenbromide is recommended for crosslinking a diaminoalkane spacer arm tocross-linked agarose. Cyanogen bromide is a well known cross-linkingagent; however, cyanate groups are formed on the agarose hydroxyl groupsnot bound to space arms or ligands.

There is evidence that all systems using cyanogen bromide for couplingresult in a significant degree of solubilization or leakage of theimmobilized ligand. Parikh and Cuatrecasas discuss the problemsassociated with cyanogen bromide in their paper "AffinityChromatography," Parikh et al., Chemical & Engineering News, Aug. 26,1985, pages 17-32. Single point attachment of the ligand can result in aleakage of 1 ppm. Leakage can be reduced but evidently not eliminated.While the level of cyanide salts is less than the lethal dose of 0.1milligrams percent, the possibility of cyanide contamination in foodproducts should be avoided.

Heterogeneous mixtures of biomolecules may also be separated bydifferential migration chromatography, in which separation is effectedby the differential migration of molecules through a filter material.The solute molecules migrate through the filter material at differentrates due to different attractions occurring between the filter materialand charges and/or functional groups on the solute molecules; the solutemolecules are not actually retained on the filter material.

U.S. Pat. No. 4,544,485 to Pinkerton et al. teaches a high-pressureliquid chromatography process in which the packing materialdiscriminates between analyte species on the basis of their differentinteractions with hydrophobic internal surfaces versus hydrophilicexternal surfaces. The hydrophobic surface may have lysine or argininecovalently bound to glyceroylpropyl groups on the support packingsurface via hydroxy functionalities. The material is useful forseparating small hydrophobic molecules (e.g., drugs) fromprotein-containing biological matrices.

U.S. Pat. No. 4,076,930 to Ellingboe teaches a column packing materialwhich may be used to separate cholesterol, among other molecules. Thematerial comprises hydroxyalkyl ethers of hydroxyalkoxy polysaccharides.Hydrocarbon radicals attached by ether linkages confer stronglylipophilic solvation characteristics.

U.S. Pat. No. 3,814,255 to Smernoff teaches a triglyceride cholesterolanalysis in which the column material comprises activated porousinorganic oxide particles.

U.S. Pat. No. 3,817,706 to Smith teaches a fluorescence quantitativethin layer chromatographic method in which an adsorbent such as alumina,silica acid or silica gel is used on a plate to separate analytesincluding cholesterol. These analytes are stained and quantified.

U.S. Pat. No. 3,997,298 to McLafferty et al. teaches a ligandchromatography-mass spectrometry system and method. Quantitative andqualitative analysis of analytes, including cholesterol, is effectedusing a system coupling a liquid chromatography column to amass-spectrometer chemical ionization detector.

Netherlands Patent Application No. 8304501A to Ultrecht teaches a columnstructure for a high-pressure liquid chromatography procedure. Steroidsand lipids may be separated.

U.S. Pat. No. 3,527,712 to Renn et al. teaches a dried agarose gel, amethod of preparation thereof and production of an aqueous agarose gel.A dissolved macro-molecular hydrocolloid is introduced into the porousstructure of an agarose gel. The hydrocolloid may include cellulosederivatives, amides or polysaccharides. The material is useful forsorting molecules having molecular weights greater than 200,000, whenpresent at concentrations of less than 5 percent. Separation of smallermolecules than molecular weight 200,000 is possible when the material ispresent at concentrations greater than 5 percent.

"Sephadex Column Chromatography as an Adjunct to Competitive ProteinBinding Assays of Steroids," Nature New Biology, 232, 21-24 (July 1971),teaches using a column packing material comprising SEPHADEX LH-20 toseparate heterogeneous mixtures of steroids. The alkylation of thehydroxyl groups of SEPHADEX makes it possible to elute with organic aswell as aqueous solvents.

"Evaluation of a High-Performance Liquid Chromatography Method forIsolation and Quantitation of Cholesterol and CholesterolEsters,"]Carroll et al., J. Lipid Res, 22(2), 359-363 (Feb. 1981),discusses using high pressure liquid chromatography for analyzingcholesterol.

Differential migration chromatographic techniques, including thoseoutlined above, provide high resolution separation of solute materials.With this procedure it is possible to separate closely related compoundsand thus enable qualitative and quantitative analysis of thesecompounds; however, such techniques are not commercially feasible forthe extraction of cholesterol from foodstuffs because too many differentsolutes would be separated, the foodstuff would be highly diluted, andpost-treatment of the foodstuff filtrate would be cumbersome.

Various potential methods for the separation of cholesterol fromfoodstuffs, including the affinity chromatography methods discussedabove, depend upon the selection of a material which has a strongaffinity for cholesterol. A number of substances are known to have suchan affinity. These include macromolecular carrier proteins, specificamino acids, specific polypeptides, and polyene antibiotics.

The most logical substances for binding cholesterol would be thosesubstances involved in cholesterol transport within biological systems.A number of papers discuss the isolation and character of thesecholesterol carrier proteins. Examples of such papers include:

In "The Role of a Carrier Protein in Cholesterol and Steroid HormoneSynthesis by Adrenal Enzymes 1, 2, " Kan et al., Biochemical andBiophysical Research Communications, 48(2), 423-429 (1972). The adrenalglands are shown to contain a sterol carrier protein (SCP) similar tothat of liver-SCP. The paper points out that SCP is required forcholesterol synthesis from squalene and steroid synthesis fromcholesterol. SCP is thought to be present in yeast and protozoa.

Takase et al., "Characterization of Sterol Carrier Protein Binding with7-Dehydrocholesterol and Vitamin D", J. Nutr. Sci. Vitaminol., 23,53-61, (1977) discusses the role of Vitamin D3 in the relationshipbetween rat liver sterol carrier protein (SCP) and cholesterol.

LeFevre et al., "Adrenal Cholesterol-Binding Protein: Properties andPartial Purification", Febs. Letters, 89(2), 287-292 (1978) discusses aheat-stable protein (CPB) present in the cytosol of adrenal glands,testes and ovaries which specifically binds tritiated cholesterol. Acase is made differentiating the CPB from the known sterol-carrierprotein present in liver.

Higuchi et al., "Comparative Studies on a Heat-StableCholesterol-Binding Protein in Dental Cyst Fluid and Serum", Int. J.Biochem., 13, 777-782 (1981) presents data indicating that dental cystfluid contains a heat-stable cholesterol-binding protein (CPB) factor. Aheated supernatant fraction of cyst fluid is reacted with a C¹⁴ labeledcholesterol. A SEPHADEX column is used to separate the bound cholesterolfrom the free cholesterol. The concentration of bound cholesterol isdetermined by plotting the radioactivity.

Chen et al., "Prostate Protein: Isolation and Characterization of thePolypeptide Components and Cholesterol Binding", J. Biol. Chem., 257(1),116-121 (1982) presents data concerning α-protein, a major protein inrat prostate secretions which originates in the rat ventral prostate.α-Protein is shown to bind cholesterol.

Sziegoleit, "Purification and Characterization of a Cholesterol-BindingProtein from Human Pancreas," Biochem. J., 207, 573-582 (1982) describesa cholesterol binding protein discovered in excreted lavage fluids.Immunologic analysis of the gut specific protein shows the organ oforigination to be the pancreas. The protein not only binds cholesterol,but also the bile salt deoxycholate. The protein comprises a singlepolypeptide chain having a molecular weight of 28,000. The isoelectricpoint is at pH 4.9.

Regenass-Klotz et al., "Specific Binding of cholesterol to ChromatinPrepared from Mouse Spleen Cells", Can. J. Biochem. Cell Biol., 62,94-99 (1984) presents data showing that cholesterol specifically bindsto the chromatin of mouse splenic lymphocytes. The evidence points tothe cholesterol actually binding to a high molecular weight protein inthe chromatin preparation and not to deoxyribonucleic acid.

The carrier proteins discussed above show great affinity for cholesteroland would theoretically provide specific ligands for affinitychromatography; the binding site of any of these proteins could beimmobilized and used for liquid chromatography to specifically removecholesterol. However, the binding site is only a small part of theprotein molecule, and thus a large mass of protein would be required toremove a small amount of cholesterol. In addition, if the naturalprotein is employed, the possibility of contaminants causing hepatitisand other viral diseases is always present. Consequently, in practicethese methods are entirely unacceptable for use in food processing.

Klimov et al., "Interaction of Cholesterol with Polypeptides and AminoAcids", documents certain binding sites on amino acids and polypeptideswhich bind cholesterol. This article teaches that amino acids andcompounds containing guanidinio groups (e.g., guanidine, metformine,arginine and polyarginine) and gamma-amino groups (e.g., lysine andpolylysine) bind to cholesterol; however, there is no suggestion forusing these substances for the extraction of cholesterol.

Certain antibiotics have been noted for their ability to bind tocholesterol. Notable amongst these are the polyenes filipin andpimaricin. Bornig et al., "Staining of Cholesterol with the FluorescentAntibiotic Filipin", Acta Histochem., 50, 110-115 (1974) documents theaffinity of filipin for non-esterified cholesterol, and cited itsutility as a histochemical stain. Patterson, "Effects of ExperimentalConditions on the Interaction of Filipin and Pimaricin withCholesterol", Antibiot. (Tokyo), 32(11), 1193-2000 (1979) documentspimaricin as having similar properties to filipin. Others have noted theaffinity that these polyenes have for cholesterol; see, for example,

Geyer et al., "Filipin --A Histochemical Fluorochrome for Cholesterol",Acta Histochem [Suppl] (Jena), 15, 207-212 (1975);

Bittman et al., "Determination of Cholesterol Asymmetry by RapidKinetics of Filipin-Cholesterol Association: Effect of Modification inLipids and Proteins", Biochemistry, 20(9), 2425-2432 (1981); and

Behnke et al., "Filipin as a Cholesterol Probe. II. Filipin CholesterolInteraction in Red Blood Cell Membranes", Eur. J. Cell Biol., 35(2),200-215 (1984). None of the references suggest the use of pimaricin orfilipin as a ligand to remove cholesterol from foodstuffs.

U.S. Pat. No. 4,297,220 to Meitzner et al. (assigned to Rohm and Haas)describes a method for adsorbing an organic material from a fluid orfluid mixture containing the same which comprises contacting the fluidor fluid mixture containing organic material with a macroreticulatedcrosslinked copolymer having a plurality of microscopic channelsresulting from liquid expulsion of a precipitating agent duringpolymerization of a monomer mixture under suspension polymerizationconditions in an aqueous media of (1) a polyvinylidene monomercontaining a plurality of ethylenically unsaturated groups in anon-conjugated relationship and (2) at least one monoethylenicallyunsaturated monomer, said copolymerization taking place in the pressure[sic] of a liquid which is a solvent for the monomer mixture and whichdoes not swell the copolymer resulting from the copolymerization, theliquid being present in an amount sufficient to cause separation of thecopolymer from the monomer phase, whereby the organic material isadsorbed by the macroreticulated crosslinked copolymer. The solvent inthe polymerization mixture causes the formation of microscopic channelsin the copolymer; the purpose of these channels appears to be tofacilitate access of the organic material to the ion-exchange centerswhich are later formed on the copolymer.

U.S. Pat. No. 4,454,056 to Kittelmann et al. describes a process for themodification of the surfaces of zeolites with organosilanes which haveat least one alkoxy group bonded to the silicon atom of the silane. Thepurpose of the surface modification is to prevent agglomeration of thezeolite when it is mixed with a detergent, so that the resultant mixturewill remain free-flowing in use.

U.S. Pat. No. 4,637,861 to Krull et al. describes a stabilized,lipid-membrane based device comprising a perturbable lipid membrane anda membrane-stabilizing support. The lipid membrane includes a complexingagent for selectively interacting with a specified chemical species toperturb the lipid membrane, and each membrane-forming lipid includes apolar head group, a first long chain through which the lipid is anchoredto a binding site on the support, and a fluidity-providing second chain,which is not attached to the support. The binding sites on the supportof spaced apart so as to provide a lipid packing density that permitsaxial rotation of each membrane-forming lipid about its long, anchoredchain, and is yet sufficiently close to provide a high ionimpermeability to the unperturbed lipid membrane, but an increasedpermeability when disturbed. The lipid is preferably anchored to thesupport via a silicon atom (see column 4, lines 35-42 of the patent).

U.S. Pat. No. 4,681,870 to Balint et al. describes a method forpreparing an immunoadsorbent material useful for removing IgG andIgG-complexes from biological fluids, this method comprising introducingfree amino or carboxyl groups on to a silica matrix, reacting the silicamatrix with purified protein A in the presence of a carbodiimide at a pHof 3.5 to 4.5 to covalently link the protein A to the silica matrixthrough the amino or carboxyl groups, and washing the silica matrix atpH 2.0 to 2.5 to remove loosely bound protein A. In effect, this methodsimply bonds the protein A, which effects the actual conjugation withthe IgG, to a support, thereby providing a solid, insoluble form of theprotein A able to function as the active material of a column throughwhich liquid to be stripped of IgG can be run.

U.S. Pat. No. 4,665,110 to Zones describes a process for the synthesisof zeolites using adamantane compounds as templating agents.

Lok et al., "The role of organic molecules in molecular sievesynthesis", Zeolites, 3, 282 (1983), is a review article discussing therelationship between the templating agents used in the synthesis ofzeolites, aluminophosphates and similar compounds, and the structures ofthe molecular sieves produced.

Lehn, "Supramolecular chemistry: receptors, catalysts and carriers",Science, 227, 849 (1985) is a general review of ways in whichpolymolecular complexes are formed, and includes a discussion of varioustypes of binding by polydentate ligands.

Wehr, "Sample preparation and column regeneration in biopolymerseparations", J. Chrom. 418, 27 (1987) discusses the use of selectivesorbents, including silica-based sorbents, for selective adsorption andfractionation of polypeptides from complex biological mixtures.

Evershed et al., "Strategy for the analysis of steryl esters from plantand animal tissues", J. Chrom. 400, 187 (1987), discusses varioustechniques for separation of steryl esters from complex biologicalmixtures, including various chromatographic separations.

Chemical Abstracts, 108, 112,170k and 150,453n, describe polydentateligands and cavitands capable of binding metal ions and small guestmolecules. There is no disclosure of separation of cholesterol or anyclosely related material.

Yamamura et al., Guest Selective Molecular Recognition by anOctadecdylsilyl Monolayer Covalently Bound on an SnO₂ Electrode, J.Chem. Soc., Chem. Commun., 1988, 79-81 discloses the technique ofadsorbing a templating molecule, such as a cholesterol derivative, on toa tin oxide surface, modifying the surface using a silane derivative,and desorbing the templating molecule in order to provide a modifiedsurface having cavities which will accommodate cholesterol or othermolecules which it is desired to adsorb. This paper demonstrates that,when the modified surface is placed in contact with a solutioncontaining cholesterol, access of Vitamin K₁ to the electrode isblocked; however, the paper does not indicate how much cholesterol isadsorbed by the modified surface, nor does it suggest that such amodified surface can be used to separate cholesterol from fluidmixtures.

Chemical Abstracts, 108, 127,097r, is an abstract of a more recent paperby the same researchers as the previous paper, and records the use ofODS-modified silica for the adsorption of artificial liposomes.

It has now been discovered that sterol compounds can be removed fromfluid mixtures by contacting the mixture with a surface-modifiedcarbonate salt. In particular, sterol compounds can be removed fromliquid comestible mixtures, including foodstuffs, in a highly selectivemanner and without substantial changes in the physical and nutritionalproperties of the foodstuff or other liquid comestible mixture bycontacting the mixture with a surface-modified carbonate salt. Also, asalready indicated, the same surface-modified carbonates can be used toremove sterol compounds from solvents, such as liquid or supercriticalcarbon dioxide, which have been used for extraction of foodstuffs, thisremoval of sterol compounds being effected without the need to evaporateand reliquify the solvent.

SUMMARY OF THE INVENTION

This invention provides a process for separation of at least one sterolcompound from a fluid mixture, this process comprising:

(a) treating a substantially insoluble carbonate salt with a firststerol compound capable of becoming adsorbed on the surface of thesubstantially insoluble carbonate salt, this treatment being effectedunder conditions effective to cause the first sterol compound to becomereversibly adsorbed on the surface of the substantially insolublecarbonate salt, thereby producing a sterol-modified carbonate;

(b) treating the sterol-modified carbonate produced in step (a) with anexcess of a surface-modifying agent, the surface-modifying agent havinga reactive group capable of reacting with the surface of thesubstantially insoluble carbonate salt, and an elongate hydrophobicportion, the treatment of the sterol-modified carbonate with thesurface-modifying agent being effected under conditions such that theadsorbed first sterol compound is not desorbed from the substantiallyinsoluble carbonate salt but substantially all of the reactive sites onthe surface of the substantially insoluble carbonate salt not covered bythe adsorbed sterol compound react with the surface-modifying agent;

(c) desorbing the first sterol compound from the substantially insolublecarbonate salt, thereby producing a surface-modified carbonate; and

(d) contacting the surface-modified carbonate from step (c) with thefluid mixture under conditions effective to permit adsorption of atleast one second sterol compound on to the surface-modified carbonate,thereby producing a fluid mixture having a reduced content of the atleast one second sterol compound.

This invention also provides a process for separation of at least onesterol compound from a fluid mixture, this process comprising contactingthe fluid mixture with a surface-modified substantially insolublecarbonate salt under conditions effective to cause selective adsorptionof the at least one sterol compound from the fluid mixture on to thesurface-modified substantially insoluble carbonate salt, thissurface-modified substantially insoluble carbonate salt having bonded toits surface a layer of elongate hydrophobic chains, this layer beinginterrupted by cavities shaped so as to selectively adsorb the at leastone sterol compound.

This invention also provides a process for separation of at least onesterol compound from a comestible material, this process comprising:

(a) treating a substantially insoluble carbonate salt with a firststerol compound capable of becoming adsorbed on the surface of thesubstantially insoluble carbonate salt, this treatment being effectedunder conditions effective to cause the first sterol compound to becomereversibly adsorbed on the surface of the substantially insolublecarbonate salt, thereby producing a sterol-modified carbonate;

(b) treating the sterol-modified carbonate produced in step (a) with anexcess of a surface-modifying agent, this surface-modifying agent havinga reactive group capable of reacting with the surface of thesubstantially insoluble carbonate salt, and an elongate hydrophobicportion, the treatment of the sterol-modified carbonate with thesurface-modifying agent being effected under conditions such that theadsorbed first sterol compound is not desorbed from the substantiallyinsoluble carbonate salt but substantially all of the reactive sites onthe surface of the substantially insoluble carbonate salt not covered bythe adsorbed sterol compound react with the surface-modifying agent;

(c) desorbing the first sterol compound from the substantially insolublecarbonate salt, thereby producing a surface-modified carbonate;

(d) contacting the comestible material with a solvent under conditionssuch that the solvent dissolves at least one sterol compound form thecomestible material, thereby producing sterol-laden solvent; and

(e) contacting the surface-modified carbonate from step (c) with thesterol-laden solvent produced in step (d) under conditions effective topermit adsorption of at least one sterol compound on to thesurface-modified carbonate, thereby producing a solvent having a reducedcontent of the at least one sterol compound.

The term "substantially insoluble carbonate salt" is used herein todenote a carbonate salt having a water solubility sufficiently low topermit the carbonate salt to react with a long chain fatty acid, such asstearic acid, with the formation of a layer of alkyl chains on thesurface of the carbonate salt, but without the dissolution of thecarbonate. Thus, the term includes not only carbonates which areregarded as truly water-insoluble (such as barium carbonate) but alsocalcium and magnesium carbonates, which are normally regarded assparingly soluble in water. However, the term excludes the alkali metalcarbonates.

The term "sterol compound" is used herein to mean cholesterol and itsderivatives, metabolites and enzymatic degradation products, and alsoincludes plant sterols and the oxidized forms of such plant sterols,provided that such compounds have a molecular shape which issufficiently similar to that of cholesterol that the sterol compoundscan be adsorbed into cavities of such a shape that they can adsorbcholesterol itself. Thus, the term "sterol compound" includes manycholesterol 3-esters, but excludes those long chain fatty acid 3-estersin which the alkyl chain is so long that the compounds are not adsorbedinto cavities which will adsorb cholesterol itself.

The "first sterol compound" used in the process of the present inventionto prepare the surface-modified adsorbent may be the same as, ordifferent form, the second sterol compound(s) which are separated formmixtures using this surface-modified adsorbent. For example, acholesterol 3-ester may be used to prepare a surface-modified adsorbent,which is then used to separate cholesterol itself form a liquid mixture.In other cases, it may be possible to use, for example, cholesterolitself to prepare a surface-modified adsorbent, and to use thissurface-modified adsorbent to remove cholesterol from a liquid mixture.

It will be apparent from the foregoing summary of this invention thatthere are two main variants of the process of the invention. In thefirst variant, the surface-modified carbonate is contacted directly withthe fluid mixture form which a sterol compound is to be removed; thisvariant will hereinafter be referred to as the "direct" process of theinvention. In the second variant, a solvent is used to remove a sterolcompound from the fluid mixture and the surface-modified carbonate isthereafter contacted with the sterol-laden solvent; this variant willhereinafter be referred to as the "indirect" process of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 of the accompanying drawings shows schematically the manner inwhich the surface-modified carbonate used in the process of the presentinvention is prepared and used to separate sterol compounds from aliquid comestible mixture; and

FIG. 2 is a highly schematic representation of an apparatus for carryingout the indirect process of the invention.

DETAILED DESCRIPTION OF THE INVENTION

As already mentioned, the present invention provides a process forseparation of a sterol compound from a fluid mixture using asurface-modified carbonate. This surface-modified carbonate is preparedby treating a carbonate with a first sterol compound capable of reactingwith the surface of the carbonate under conditions effective to causethe sterol compound to become reversibly adsorbed on the surface of thecarbonate, thereby producing a sterol-modified carbonate on to thesurface of which the first sterol compound is reversibly adsorbed, asillustrated at B in the accompanying FIG. 1, in which "STER" representsa sterol compound.

The sterol-modified carbonate thus produced is then treated with anexcess of a surface-modifying agent, which has both a reactive groupcapable of reacting with the surface of the carbonate, and an elongatehydrophobic portion, so that the sterol compound stays adsorbed on thecarbonate but substantially all of the reactive sites on the surface ofthe carbonate not covered by the adsorbed sterol compound react with thesurface-modifying agent. The resultant product is shown schematically atC in FIG. 1, from which it will be seen that the adsorbed molecules ofthe sterol compound (which in practice are separated from one another byseveral angstroms), are each surrounded by the elongate hydrophobicchains of the surface-modifying agent. It should be noted that, incontrast to the sterol compound, which is only adsorbed on to thesurface of the carbonate, the surface-modifying agent needs to be firmlybonded to the carbonate surface, normally by means of covalent and/orionic bonds. In practice, the density of the surface-modifying agent onthe carbonate surface is such that the hydrophobic chains areclose-packed, producing an organized and immobilized two-dimensionalphase surrounding the molecules of sterol compound, so that thesurface-modifying agent and the sterol compound in effect form atwo-dimension clathrate.

Next, the first sterol compound is desorbed from the carbonate, therebyproducing a surface-modified carbonate, as illustrated at D in FIG. 1.Such desorption is typically achieved by contacting the carbonate with asolvent in which the first sterol compound is freely soluble. Thedesorption of the sterol compound leaves the surface-modified carbonatewith an organized and immobilized two-dimensional phase surrounding"holes" or vacancies which are especially shaped to fit the sterolcompound which has been removed therefrom. (And since the sterolcompounds used in the process of the present invention are chosen tohave a molecular shape similar to that of cholesterol itself, thesevacancies will also be especially shaped to fit cholesterol and similarmolecules.) Hence, when the surface-modified carbonate is placed incontact with a sterol-containing liquid, such as a foodstuff, thesurface-modified carbonate adsorbs sterol compounds (designated "CHOL"in FIG. 1) in a highly selective manner, since only cholesterol or amolecule having a very similar size and shape is capable of entering thevacancies left behind when the sterol compound is desorbed. The processis analogous to that by which molecular sieves can adsorb molecules of aparticular size and shape from complex mixtures, and indeed thesurface-modified carbonate may be regarded as analogous to atwo-dimensional molecular sieve.

(It will be apparent to those skilled in thermodynamics that inpractice, the hydrophobic chains on the surface-modified carbonate areunlikely to retain their original positions exactly, as shown at D inFIG. 1, since entropy considerations would suggest that the hydrophobicchains would tend to enter into the vacancies. However, since the chainsare bound to the surfaces at their bases, even if the chains do tend toenter the vacancies, free energy considerations will be such that the"wandering" chains will readily leave the vacancies when a sterolmolecule is available for adsorption therein.)

Thus, exposing the surface-modified carbonate to a sterol-containingfluid mixture results in highly selective adsorption of sterol compoundsinto the vacancies on the carbonate surface, leaving the fluid mixturedepleted in sterol compounds. The sterol-depleted mixture can then bephysically separated from the sterol-laden carbonate, and the sterolcompounds removed from the carbonate by contacting the carbonate with asolvent in which the sterol compounds are readily soluble.

When the surface-modified carbonate is to come into direct contact withfood materials (as, for example, when the direct method of the presentinvention is to be used to reduce the sterol level in egg yolks), caremust of course be taken to ensure that the surface-modified carbonatedoes not introduce contaminants into the food material. Under thesecircumstances, the preferred carbonates for use in the present processare calcium and magnesium carbonates (including mixed calcium/magnesiumcarbonates such as dolomite); calcium carbonate is the especiallypreferred carbonate. Both calcium and magnesium carbonates are generallyrecognized as safe for use in foodstuffs, and indeed calcium carbonateis often added to doughs and other food materials as a nutritionalsupplement for use in the diet; thus grades of calcium carbonatesuitable for contact with food materials are readily availablecommercially.

Obviously, the indirect process of the present invention, in which thesurface-modified carbonate is used to remove sterol compounds from asterol-laden solvent, imposes fewer restrictions upon the nature of thecarbonate since, for example, a carbonate which is not itself acceptablefor direct contact with food may be acceptable for us in the indirectprocess provided that the carbonate does not introduce unacceptablecontaminants into the solvent.

The carbonate used to prepare the surface-modified carbonate isdesirably in pulverulent and/or precipitated form. Since the adsorptionof sterol compounds is a surface phenomenon, a finely divided carbonateshould be employed so as to provide a high surface area available foradsorption per unit weight of the carbonate. However, the carbonateshould not be so finely divided that, after the carbonate has beenloaded with sterol compounds by contact with the fluid mixture,difficulty is encountered in separating the carbonate from thesterol-depleted mixture by, for example, filtration or centrifugation.Grades of calcium carbonate less than 200 U.S. mesh are commerciallyavailable and should generally be acceptable for use in the presentprocess. As those skilled in adsorption technology are aware, theavailable surface area of a carbonate proposed for use in the presentprocess may readily be determined by conventional tests, such as theBrunauer-Emmett-Teller test using nitrogen at -183° C.

The carbonate employed in the process of the present invention must bein a form which can adsorb the first sterol compound employed to producethe surface-modified carbonate, and which can react with thesurface-modifying agent. Since the surface-modifying agents react withcarbonate or bicarbonate groups on the carbonate surface, the carbonateshould be in a form in which its surface carbonate groups are readilyavailable for reaction. In addition, the carbonate should be free fromsurface contaminants which might interfere with the adsorption of thesterol compound or reaction with the surface-modifying compounds; thosefamiliar with the treatment of adsorbents will be familiar with methods,such as vacuum degassing or washing, which may be employed to ensurethat the carbonate is in a proper state for use in the present process.Finally, if the carbonate is to be employed in the direct process of theinvention, in which the surface-modified carbonate comes into intimatecontact with the fluid mixture, it is of course essential that thecarbonate be free from any material, for example heavy metals, whichcould leach into and be unacceptable in the treated mixture.

Preferred sterol compounds for use in the present process arecholesterol and its 3-esters, preferably esters of dibasic acids, andmost desirably carbonate esters, for example 2-cholesteryl3,6-dioxadecyl carbonate. A specific preparation of this ester is givenin Example 1 below. Since the sterol compounds are usually solids, theywill normally be dissolved in a solvent before being contacted with thecarbonate; appropriate solvents for this purpose are alkanes, forexample heptane. The solvent used is preferably not one in which thesterol compound is highly soluble, since if the sterol compound is toosoluble, it may be highly solvated and be adsorbed by the carbonatesurface only with difficulty. For example, 3-cholesteryl 3,6-dioxadecylcarbonate is highly soluble in diethyl ether, which makes an excellentsolvent for desorption of the compound from the carbonate, but use ofdiethyl ether is not recommended for adsorption of the compound on tothe carbonate.

In order to ensure that the surface-modified carbonate has a highadsorption of, and a high selectivity for, sterol compounds, the amountof first sterol compound placed on the carbonate should be carefullycontrolled. If too little sterol compound is placed on the surface, onlya limited number of vacancies will be created on the carbonate, and thusthe surface-modified carbonate will only adsorb a small amount of sterolcompounds per unit weight of carbonate. On the other hand, if too muchsterol compound is employed, the surface of the carbonate may becomelargely or completely covered with the sterol compound. In the formercase, since sterol compound molecules will be adsorbed close together,treatment of the sterol-modified carbonate with the surface-modifyingagent will result in the formation of vacancies which do not have theshape of a single molecule of the sterol compound, and which will thusadsorb molecules other than sterol compounds. In the latter case, novacancies may be formed at all.

The optimum amount of first sterol compound for treatment of a specificcarbonate may be determined by routine empirical tests, which willpresent no difficulty to those skilled in adsorption technology. Ingeneral, it has been found that use of a quantity of first sterolcompound which provides about one molecule of the compound per 100 Å² ofthe surface of the carbonate (as determined by the conventionalBrunauer-Emmett-Teller test using nitrogen at -183° C.) gives goodresults.

As is well known to those skilled in adsorption technology, forthermodynamic reasons the maximum amount of adsorbate per unit surfacearea of carbonate normally decreases with increasing temperature.Accordingly, during the adsorption process, the temperature is desirablykept low; in practice, operating at room temperature (about 20° C.) hasbeen found to give satisfactory results.

Once the carbonate surface has been loaded with the desired amount offirst sterol compound, the surface is contacted with thesurface-modifying agent. Preferred surface-modifying agents for use inthe process of the present invention are fatty acids, preferably thosecontaining at least ten, and preferably from about 14 to about 22,carbon atoms. The fatty acid alternatively may contain from about 16 to18 carbon atoms. Specific acids suitable for use in the process include,for example, myristic, palmitic, stearic, arachidonic, palmitoleic,oleic, linoleic and linolenic acids. These fatty acids and theirnon-natural counterparts having odd numbers of carbon atoms, react withcarbonate or bicarbonate groups present on a carbonate surface withelimination of carbon dioxide and/or bicarbonate ion and formation of anRCO₂ residue. These residues bind to the carbonate, while leaving thehydrophobic "tail" of the fatty acid surface-modifying agent free toextend away from the carbonate surface and produce the organized andimmobilized two-dimensional phase surrounding the molecules of sterolcompound.

For obvious reasons, it is desirable to ensure that the first sterolcompound is not desorbed from the carbonate surface while thesterol-modified carbonate is being reacted with surface-modifying agent.Thus, the surface-modifying agent should not be added to the carbonatein the form of a solution containing a large amount of a solvent inwhich the sterol compound is freely soluble, lest a substantial part ofthe adsorbed sterol compound be desorbed by this solvent. Provided thatthe sterol compound and the surface-modifying agent will not react withone another and are soluble in the same types of solvents, desorption ofthe sterol compound may be minimized by simply adding the solution ofthe surface-modifying agent directly to the mixture of the carbonate andthe solution of the sterol compound. In addition, it is desirable tominimize the time for which the surface-modifying agent is contactedwith the sterol-modified carbonate; with calcium carbonate and a fattyacid surface-modifying agent, a contact time of about 5 to about 10minutes should give good results. Conveniently, fatty acidsurface-modifying agents are added to the sterol-modified carbonate assolutions in aqueous alkanols, for example aqueous propanol.

The amount of the surface-modifying agent added to the sterol-modifiedcarbonate must be sufficient to allow substantially all of the reactivesites on the surface of the carbonate not covered by the adsorbed sterolcompound to react with the surface-modifying agent, in order that thehydrophobic chains of the surface-modifying agent may completely coverthe surface of the carbonate and surround the adsorbed sterol compound,thereby forming, after desorption of the sterol compound, vacancieswell-defined to adsorb only sterol compounds; incomplete coverage of thesurface of the carbonate will leave the vacancies ill-defined, so thatthe carbonate will thereafter not adsorb sterol compounds effectively,or the adsorption will lack the necessary selectivity. Since thesurface-modifying agent reacts only with the surface of the carbonate,the necessary amount of surface-modifying agent per unit weight ofcarbonate varies with the surface area per unit weight of the carbonate.However, the minimum amount of surface-modifying agent required in anyparticular case is readily determined by routine empirical tests, itbeing only necessary to determine the minimum quantity which willproduce maximum sterol compound adsorption. It should be noted that,because of the irregular nature of the surface of carbonates at theatomic level, the available surface area of the carbonate varies withthe size of the molecule being adsorbed. This gives rise to theobservation of an apparent surface area (for the surface-modifyingagents) which is less than the actual surface area as measured byphysical tests. Because of the difference between the molecular sizes ofthe nitrogen normally used in the BET test and the surface-modifyingagents used in the present process, the difference is available surfacearea can become very pronounced; for example, a carbonate having a BETsurface area of 500 m² /g might have an available surface area of onlyabout 200 m² /g when measured by gravimetric methods using a fatty acidsurface-modifying agent. Consequently, it is not desirable to calculatethe amount of surface-modifying agent required by dividing the normalBET surface area by the molecular cross-sectional area of a molecule ofthe surface-modifying agent.

After the treatment of the carbonate with the surface-modifying agenthas been completed, the first sterol compound must be desorbed from thecarbonate surface. This desorption is readily effected by contacting thesurface with a solvent in which the sterol compound is readily soluble;cholesterol and most of its derivatives used as sterol compounds in theprocess of the present invention are readily soluble in non-polarsolvents, especially diethyl ether, and such solvents are thereforerecommended for desorption of these cholesterol compounds. In manycases, desorption with diethyl ether or a similar solvent recovers thesterol compound almost quantitatively, thereby permitting the expensivesterol compound to be used in the treatment of further batches ofcarbonate.

The surface-modified carbonate thus produced may then be employed toremove sterol compounds from comestible liquid and other fluid mixtures.The "comestible liquid mixtures" used in the process of the presentinvention include not only foodstuffs, for example egg yolk, which areinherently liquid, but also solid or semi-solid foodstuffs, for examplesolid animal fats such as lard, which can be made in a liquid form bydissolving them in an appropriate solvent which does not interfere withthe sterol-removal process. The same surface-modified carbonate can beused to remove sterol compounds from sterol-laden solvents, such assupercritical carbon dioxide.

To ensure maximum removal of sterol compounds from the fluid mixture, itis of course necessary to ensure that the mixture is intimatelycontacted with the surface-modified carbonate. Although, at least intheory, the necessary intimate contact could be achieved by passing thefluid mixture through a column of the surface-modified carbonate, sothat the surface-modified carbonate forms the stationery phase of thesystem, in practice such a column separation process is undesirable formany fluid mixtures, especially foodstuffs. Many foodstuffs, such as eggyolk, are proteinaceous and highly viscous. The pressures necessary toforce such viscous liquids through columns of surface-modified carbonateat the rates required for practice of this invention on a commercialscale are high, and consequently, the fluid mixture will be exposed tolarge shear forces as it passes through the column. Such large shearforces may have undesirable effects upon the proteins present in thefluid.

To avoid such problems in column treatment, as already mentioned it isnormally preferred to carry out the process of the present inventionwith the surface-modified carbonate in pulverulent form, thoroughlyadmixing this pulverulent carbonate with the fluid mixture, and thenseparating the sterol-laden carbonate from the sterol-reduced fluidmixture by, for example, filtration or centrifugation. The separatedsterol-laden carbonate can then have the sterol compounds desorbedtherefrom in a manner similar to that in which the first sterol compoundis desorbed during preparation of the surface-modified carbonate (forexample, by contacting the sterol-laden carbonate with diethyl ether),and the surface-modified carbonate recycled for use in treating furtherbatches of fluid mixture. If desired, the sterol compounds can berecovered from the diethyl ether or other solvent.

The following Examples are now given, though by way of illustrationonly, to show details of reagents, conditions and techniques which mightbe employed in the process of the present invention. Unless otherwisestated, all parts are by weight.

EXAMPLE 1 Synthesis of 3-cholesteryl 3,6-dioxadecyl carbonate

This Example illustrates the synthesis of a sterol compound which may beuseful in the process of the present invention.

A solution of 4.49 g. (0.010 mole) of cholesteryl chloroformate in aminimum amount of chloroform was added to a solution of 1.62 g (0.01mole) of 2(2-butoxyethoxy)ethanol and 1.2 g (0.015 mole) of pyridine in20 mL of chloroform and the resultant solution stirred at ambienttemperature for 12 hours. The solution was then washed with 5 percenthydrochloric acid and with water and dried over anhydrous sodiumsulfate. Filtration, evaporation, and chromatography of the residue oversilica gel (12:1, hexane:ethyl acetate eluent) afforded a pure sample ofthe sterol compound, 3-cholesteryl 3,6-dioxadecyl carbonate as a clear,viscous liquid.

EXAMPLE 2 Adsorption of Sterol Compound on Calcium Carbonate

This example illustrates the adsorption of a sterol compound, namelycholesterol, on calcium carbonate.

Calcium carbonate, 98 percent through 200 U.S. mesh, is added to asolution of cholesterol in heptane and the resultant slurry is stirredfor 12 hours; the amount of cholesterol used is calculated to give 1molecule of cholesterol per 100 Å² of surface of the calcium carbonate.The adsorption of the cholesterol on to the carbonate may be followed bymonitoring the UV absorption of the solution, which decreases as thecholesterol is adsorbed. It will be found that the cholesterol readilyabsorbs from heptane on to the virgin carbonate surface to producesub-monolayer coverage.

EXAMPLE 3 Surface Modification of Calcium Carbonate with Stearic acid

This Example illustrates the reaction of the sterol-modified carbonateproduced in Example 2 with a surface-modifying agent.

The slurry of sterol-modified calcium carbonate produced in Example 2above is mixed with a solution of stearic acid in a minimum amount of50:50 w/w propanol/water mixture. After stirring for ten minutes, theresultant solid is allowed to settle, the liquid phase decanted, and theremaining solid washed with fresh aqueous propanol. Filtration andsubsequent vacuum drying afforded the sterol-laden surface-modifiedcarbonate product as a fine white powder.

EXAMPLE 4 Desorption of Sterol Compound and Formation of FinalSurface-modified Calcium Carbonate

This Example illustrates desorption of the sterol compound from thesterol-laden surface-modified carbonate produced in Example 3 above toproduce the final surface-modified calcium carbonate havingsterol-removal capacity.

1 G. of the white powder prepared in Example 3 above is added to 20 mLof diethyl ether and the resultant slurry is warmed to 35° C. for 30minutes. The liquid phase remaining after the heating is removed bymeans of a pipet and evaporated to dryness. The resultant residue isthen analyzed to determine the amount of cholesterol present. It will befound that the amount of desorbed cholesterol is nearly equal to thatoriginally used to treat the starting calcium carbonate. Since thecholesterol is recovered unchanged, it may be reused to treat furtherbatches of calcium carbonate, thereby serving as a catalytic agent forthe preparation the surface-modified calcium carbonate.

EXAMPLE 5 Removal of Cholesterol from Mixture by Surface-ModifiedCalcium Carbonate

This Example demonstrates the sterol adsorption capacity of thesurface-modified calcium carbonate produced in Example 4 above.

A 1 g. sample of the surface-modified calcium carbonate prepared inExample 4 above is stirred for 18 hours with 20 mL of a heptane solutioncontaining 18 mM of cholesterol. The solid is recovered by filtration,dried under vacuum, and analyzed to assay for cholesterol. Thecholesterol loading determined by this procedure is typically in excessof 0.1 grams of cholesterol per gram of calcium carbonate.

This Example, together with Example 4, shows that cholesterol isrecognized by the surface-modified calcium carbonate, and can beadsorbed and desorbed by simple procedures. Since cholesterol is notadsorbed by a calcium carbonate surface which has been completelycovered with alkyl chains, it follows that the cholesterol is notadsorbed on the network of alkyl chains which cover much of the calciumcarbonate surface, but rather occurs at the special shape-recognizingsites created by the original sterol compound molecules. Since theoriginal sterol compound molecules could be removed to produce thesurface-modified calcium carbonate in a form ready for adsorption ofcholesterol or other sterol compounds, it follows that thesurface-modified calcium carbonate has good stability and can be usedrepeatedly in a cyclic process to remove sterol compounds from fluidmixtures.

EXAMPLE 6 Removal of Cholesterol from Supercritical Carbon Dioxide

This Example illustrates schematically the way in which thesurface-modified calcium carbonate produced in Example 4 above could beused to remove sterol compounds from supercritical carbon dioxide, whichhas itself been used to extract sterol compounds from a foodstuff.

The apparatus used in this process is shown in a highly schematic mannerin FIG. 2 of the accompanying drawings. In this Figure, the foodstuff tobe treated is place in a vessel 10 provided with an inlet line 12 and anoutlet line 14. Supercritical carbon dioxide is pumped by means of apump 16 from the inlet line 12 through the vessel 10 and into the outletline 14.

In practice, as for example when the treatment of the foodstuff iseffected in the manner described in the aforementioned U.S. Pat. No.4,692,280, the apparatus used to contact the carbon dioxide with thefoodstuff may be considerably more complicated than a simple vessel 10and may include numerous interconnected vessels, lines, etc. However, inso far as the modification of such a process effected by the presentinvention is concerned, any process for treatment of a foodstuff withsupercritical carbon dioxide may conceptually be regarded as takingplace in a closed vessel which receives "clean" carbon dioxide at aninlet and expels sterol-laden carbon dioxide at an outlet.

The outlet line 14 is connected to a two-way valve 18, which has outletsconnected via lines 20 and 22 to recovery vessels 24 and 26respectively, these vessels both being filled with the surface-modifiedcalcium carbonate produced in Example 4 above. These vessels 24 and 26each have an outlet connected via a line 28 or 30 to the inlet of thepump 16.

To make up the inevitable small losses of carbon dioxide, the apparatusis provided with a carbon dioxide reservoir 32, which is connected via avalve 34 to the line 12.

A reservoir 36 is connected via a line 38 provided with a pump 39 to atwo-way valve 40, the outlets of which are connected via lines 42 and 44to the recovery vessels 24 and 26 respectively. The recovery vesselshave outlets connected via lines 46 and 48 respectively, provided withcheck valves 50 and 52 respectively, to a waste solvent collectionvessel 54.

The apparatus shown in FIG. 2 is used in the following manner. Thefoodstuff to be treated is placed in the vessel 10 and supercriticalcarbon dioxide is circulated through the foodstuff by means of the pump16. Sterol-laden carbon dioxide leaves the vessel 10 via the outlet line14, and then this sterol-laden carbon dioxide reaches the valve 18, itis initially routed via the line 20 to the recovery vessel 24. As thecarbon dioxide passes through the vessel 24, most of the sterolcompounds in the carbon dioxide are adsorbed by the surface-modifiedcalcium carbonate in the vessel 24, so that substantially sterol-freecarbon dioxide leaves the vessel 24 via the line 28, and is recirculatedby the pump 16 back to the vessel 12.

This process continues (in normal practice, with occasional replacementof the foodstuff in the vessel 10 with further batches of foodstuff)until the surface-modified calcium carbonate in the vessel 24 is ladenwith almost its maximum amount of sterol compounds. At this time, thevalve 18 is shifted to its other position, so that sterol-laden carbondioxide from the line 14 is routed via the line 22, the recovery vessel26 (where most of the sterol compounds are removed), the line 30 and thepump 16 back to the vessel 10.

To remove the sterol compounds from the surface-modified calciumcarbonate in the vessel 24 and thus prepare the calcium carbonate forreuse, the vessel 36 is filled with diethyl ether or another appropriatesolvent and this solvent is pumped by the pump 39 along the line 38 tothe valve 40, which is set to route the ether along the line 42 to therecovery vessel 24. The ether desorbs the sterol compounds from thecalcium carbonate in the vessel 24, and the resulting sterol-laden etherpasses along the line 46, though the check valve 50 and into the wastereservoir 54. (Obviously, if diethyl ether or another highly inflammablesolvent is being used, appropriate precautions should be taken to ensurethat the ether in the reservoir 54 does not ignite.) The check valve 52serves to prevent back-flow of the ether into the recovery vessel 26. Ifdesired, the sterol-laden ether in the reservoir 54 may be distilled orotherwise treated to remove sterol compounds therefrom, therebypermitting reuse of the ether.

When the surface-modified calcium carbonate in the vessel 26 becomesladen with almost its maximum amount of sterol compounds, the valve 18is again shifted to route the carbon dioxide from the line 14 to thevessel 24, and the valve 40 is shifted to its other position, so thatthe ether passes through the vessel 26 and desorbs the sterol compoundsfrom the calcium carbonate in that vessel.

From the foregoing, it will be seen that the apparatus shown in FIG. 2allows continuous extraction of sterol compounds from foodstuffs usingsupercritical carbon dioxide without the need for evaporation andrecondensation of the carbon dioxide between passes through thefoodstuff. Accordingly, the process of the present invention justdescribed with reference to FIG. 2 incurs much lower energy costs thanconventional processes employing supercritical carbon dioxide to removesterol compounds from foodstuffs.

Processes generally similar to those of the present invention but usingadsorbents other than carbonates are described and claimed in anotherapplication by the present inventors, of even date herewith, andentitled "Process for separation of sterol compounds from fluidmixtures".

What is claimed is:
 1. A process for separation of at least one sterolcompound from a fluid mixture, said process comprising:(a) treating asubstantially insoluble carbonate salt with a first sterol compoundcapable of become adsorbed on the surface of the substantially insolublecarbonate salt, said treatment being effected under conditions effectiveto cause the first sterol compound to become reversibly adsorbed on thesurface of the substantially insoluble carbonate salt, thereby producinga sterol-modified carbonate; (b) treating the sterol-modified carbonateproduced in step (a) with an excess of a surface-modifying agent, thesurface-modifying agent having a reactive group capable of reacting withthe surface of the substantially insoluble carbonate salt, and anelongate hydrophobic portion, the treatment of the sterol-modifiedcarbonate with the surface-modifying agent being effected underconditions such that the adsorbed first sterol compound is not desorbedfrom the substantially insoluble carbonate salt but substantially all ofthe reactive sites on the surface of the substantially insolublecarbonate salt not covered by the adsorbed sterol compound react withthe surface-modifying agent; (c) desorbing the first sterol compoundfrom the substantially insoluble carbonate salt, thereby producing asurface-modified carbonate; and (d) contacting the surface-modifiedcarbonate from step (c) with the fluid mixture under conditionseffective to permit adsorption of at least one second sterol compound onto the surface-modified carbonate, and separating the surface-modifiedcarbonate from the fluid mixture thereby producing a fluid mixturehaving a reduced content of the at least one second sterol compound. 2.A process according to claim 1 wherein the fluid mixture is a liquidcomestible mixture comprising cholesterol and at least one protein.
 3. Aprocess according to claim 2 wherein the liquid comestible mixturecomprises egg yolk.
 4. A process according to claim 1 wherein the firststerol compound is cholesterol itself or a derivative thereof.
 5. Aprocess according to claim 4 wherein the first sterol compound is acholesterol 3-ester.
 6. A process according to claim 4 wherein the firststerol compound is cholesterol itself.
 7. A process according to claim 1wherein the surface-modifying agent is a fatty acid containing at leastten carbon atoms.
 8. A process according to claim 7 wherein thesurface-modifying agent is selected from the group consisting ofmyristic, palmitic, stearic, arachidonic, palmitoleic, oleic, linoleiclinolenic acids and mixtures thereof.
 9. A process according to claim 1wherein, in step (c), the desorption of the first sterol compound fromthe substantially insoluble carbonate salt is effected by contacting thecarbonate with a solvent in which the first sterol compound is soluble.10. A process according to claim 1 wherein, following step (d), thefluid mixture having a reduced content of the at least one second sterolcompound is separated from the surface-modified carbonate, thesurface-modified carbonate is treated to remove at least part of thesterol compound(s) therefrom, and the treated surface-modified carbonateis thereafter contacted with a second portion of the fluid mixture. 11.A process to claim 10 wherein the surface-modified carbonate is inpulverulent, or precipitated form and is mixed with the fluid mixture,and the resultant mixture of fluid mixture and carbonate is thereafterseparated by filtration or centrifugation.
 12. A process according toclaim 1 wherein the substantially insoluble carbonate salt is selectedfrom the group consisting of calcium carbonate, magnesium carbonate andmixtures thereof.
 13. A process according to claim 12 wherein thecarbonate is calcium carbonate.
 14. A process according to claim 12wherein the first sterol compound is cholesterol itself.
 15. A processaccording to claim 12 wherein the surface-modifying agent is a fattyacid containing at least ten carbon atoms.
 16. A process according toclaim 15 wherein the surface-modifying agent is selected from the groupconsisting of myristic, palmitic, stearic, arachidonic, palmitoleic,linoleic, linolenic acids and mixtures thereof.
 17. A process accordingto claim 12 wherein, following step (d), the fluid mixture having areduced content of the at least one second sterol compound is separatedfrom the surface-modified carbonate, the surface-modified carbonate istreated to remove at least part of the sterol compound(s) therefrom, andthe treated surface-modified carbonate is thereafter contacted with asecond portion of the fluid mixture.
 18. A process according to claim 12wherein the surface-modified carbonate is in pulverulent, orprecipitated form and is mixed with fluid mixture, and the resultantmixture of fluid mixture and carbonate is thereafter separated byfiltration or centrifugation.
 19. A process according to claim 12wherein, in step (b), the surface-modifying agent is added to thecarbonate in the form of a solution in an aqueous alcohol.
 20. A processaccording to claim 19 wherein the aqueous alcohol is aqueous propanol.21. A process according to claim 1 wherein the fluid mixture comprises asolvent which has been contacted with a sterol-containing foodstuff. 22.A process according to claim 21 wherein the solvent comprises liquid orsupercritical carbon dioxide.
 23. A process for separation of at leastone sterol compound from a fluid mixture, said process comprisingcontacting the fluid mixture with a surface-modified substantiallyinsoluble carbonate salt under conditions effective to cause selectiveadsorption of the at least one sterol compound from the fluid mixture onto the surface-modified substantially insoluble carbonate salt, saidsurface-modified substantially insoluble carbonate salt having bonded toits surface a layer of elongate hydrophobic chains, said layer beinginterrupted by cavities shaped so as to selectively adsorb the at leastone sterol compound and separating the surface-modified carbonate saltfrom the fluid mixture.
 24. A process according to claim 23 wherein thesurface-modified carbonate is selected from the group consisting of asurface-modified calcium carbonate, surface-modified magnesium carbonateand mixtures thereof.
 25. A process according to claim 23 wherein saidhydrophobic chains are bonded to the surface of the carbonate via --CO₂groups.
 26. A process according to claim 25 wherein said hydrophobicchains are alkyl groups each containing at least ten carbon atoms, andeach alkyl group has attached to one end thereof a --CO₂ group, thecarbon atom of said --CO₂ group being bonded to the surface of thecarbonate.
 27. A process according to claim 26 wherein the alkyl groupscontain from about 14 to about 22 carbon atoms.
 28. A process accordingto claim 27 wherein the alkyl groups contain from about 16 to about 18carbon atoms.
 29. A process according to claim 23 wherein the fluidmixture comprises cholesterol and at least one protein.
 30. A processaccording to claim 29 wherein the fluid mixture comprises egg yolk. 31.A process according to claim 23 wherein the fluid mixture comprisesliquid or supercritical carbon dioxide.
 32. A process for separation ofat least one sterol compound from a combustible material, said processcomprising:(a) treating a substantially insoluble carbonate salt with afirst sterol compound of becoming adsorbed on the surface of thesubstantially insoluble carbonate salt, said treatment being effectedunder conditions effective to cause the first sterol compound to becomereversibly adsorbed on the surface of the substantially insolublecarbonate salt, thereby producing a sterol-modified carbonate; (b)treating the sterol-modified carbonate produced in step (a) with anexcess of a surface-modifying agent, said surface-modifying agent havinga reactive group capable of reacting with the surface of thesubstantially insoluble carbonate salt, and an elongate hydrophobicportion, the treatment of the sterol-modified carbonate with thesurface-modifying agent being effected under conditions such that theadsorbed first sterol compound is not desorbed from the substantiallyinsoluble carbonate salt but substantially all of the reactive sites onthe surface of the substantially insoluble carbonate salt not covered bythe adsorbed sterol compound react with the surface-modifying agent; (c)desorbing the first sterol compound from the substantially insolublecarbonate salt, thereby producing a surface-modified carbonate; (d)contacting the comestible material with a solvent under conditions suchthat the solvent dissolves at least one sterol compound from thecomestible material and separating the surface-modified carbonate fromthe comestible material, thereby producing sterol-laden solvent; and (e)contacting the surface-modified carbonate from step (c) with thesterol-laden solvent produced in step (d) under conditions effective topermit adsorption of at least one sterol compound on to thesurface-modified carbonate, thereby producing a solvent having a reducedcontent of the at least one sterol compound.
 33. A process according toclaim 32 wherein the solvent having a reduced content of the at leastone sterol compound produced in step (e) is thereafter contacted with asecond portion of the comestible material under conditions such that thesolvent dissolves at least one sterol compound from the comestiblematerial.
 34. A process according to claim 32 wherein the solventcomprises liquid or supercritical carbon dioxide.
 35. A processaccording to claim 32 wherein the substantially insoluble carbonate saltis selected from the group consisting of a calcium carbonate, magnesiumcarbonate and mixtures thereof.
 36. A process according to claim 35wherein the substantially insoluble carbonate salt is calcium carbonate.37. A process according to claim 32 wherein the comestible materialcomprises egg yolk or fish oil.